Antineoplastic conjugates of transferin, albumin and polyethylene glycol

ABSTRACT

Conjugates of transferrin, albumin and polyethylence glycol consisting of native or thiolated transferrin or albumin or of polyethylene glycol (MW between approximately 5,000 and 20,0000) with at least one HS—, HO— or H 2 N group and cytostatic compounds derived through maleinimide or N-hydroxysuccinimide ester compounds, such as doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxandrone, chloroambucil, melphalan, 5-fluorouracyl, 5′-desoxy-5-fluorouridine, thioguanine, methotrexate, paclitaxel, docetaxel, topotecan, 9-aminocamptothecin, etoposide, teniposide, mitopodoside, vinblastine, vincristine, vindesine, vinorelbine or a compound of general formula A, B, C or D, where n=0-6, X=—NH 2 , —OH, —COOH, —O—CO—R—COR*, —NH—CO—R—COR*, where R is an aliphatic carbon chain with 1-6 carbon atoms or a substituted or unsubstituted phenylene group and R*H, phenyl, alkyl with 1-6 carbon atoms.

This application is a continuation of U.S. application Ser. No.09/254,598, filed on May 21, 1999, now U.S. Pat. No. 6,310,039, whichwas a national stage filing under 35 U.S.C. §371 of InternationalApplication No. PCT/DE97/02000 filed on Sep. 9, 1997, whichInternational Application was published by the International Bureau inon Mar. 19, 1998.

FIELD OF THE INVENTION

This invention relates to tumor-inhibiting conjugates of proteins andpolymers consisting of a suitable carrier system and cytostaticcompounds. Further, the invention relates to methods for the productionof such conjugates and the use of these. Immuno-conjugates or conjugatesof protein or of polymer are compounds which consist of a suitablecarrier substance, such as, for example, an antibody, a growth factor, astructure similar to hormones or peptides, a protein or a polymer, andone or more cytotoxic active substances such as, for example,cytostatics, toxins or radioactive isotopes. The carrier substanceshave, as a rule, the characteristic of preferably accumulating in thetumor tissue, so that in this way also the active substance bound to thecarrier substance accumulates in the tumor tissue and thus a selectiveanti-tumor therapy is achieved. Chemoimmuno-conjugates are conjugates ofcarrier substances and cytostatic compounds, wherein the carrier, as arule, is an antibody.

PRIOR ART

The cytostatics currently used against cancers have a series of strongsystemic side-effects and do not exhibit accumulation in the tumortissue, so that new derivatives and formulations are being researchedwhich make selective anti-tumor therapy possible. For this purpose.chemoimmuno-conjugates or conjugates of proteins or of polymersconsisting of one suitable carrier substance or cytostatics are beingdeveloped.

As carrier substances, among others, antibodies, growth factors, serumproteins, structures similar to hormones or peptides, or polymers areconsidered, for which, as a rule, an accumulation in the tumor tissue isknown (Mägerstädt, M.: Antibody Conjugates and Malignant Disease,Library of Congress 1990: Chadwick, C. M.: Receptors in Tumour Biology,Cambridge University Press, 1984, Seymour, L. W. CRC Crit. Rev. Ther.Drug Carrier Sys. (1992), 9, 135-187; Maeda, H.; Matsumura, Y. CRC Crit.Rev. Ther. Drug Carrier Sys. (1989), 6, 193-210). The present inventioncomprises human serum transferrin and serum albumin as carrier proteins,of which the accumulation in the tumor tissue is documented (Ward, S. G.Taylor, R. C.: 1-54, in Metal-Based Drugs (Gielen, M. F. (Ed.)), FreundPublishing House Ltd, 1988; Sinn, H., Schrenk, H. H., Friedrich, A.,Schilling, U. and Maier-Borst, W. (1990), Nucl. Med Biol. Vol. 17(8),819-827) as well as polyethylene glycols (PEGs) as carriers ofcytostatic compounds (Topchieva, I. N. (1990), Polym. Sci. USSR 32,833-851; Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications (1992), Ed. J. M. Harris, Plenum Press, New York). PEGsare, due to their bio-compatibility, their good water-solubility andsynthetic divergence, very suitable for the development of therapeuticpolymer conjugates. In recent years, PEGs have been conjugated mainlywith medically significant proteins and enzymes (Overview in Topchieva,I. N. (1990), Polym. Sci. USSR, 32, 833-851). The production ofchemoimmuno-conjugates and conjugates of proteins or of polymers occursgenerally either through direct coupling of carrier substance and activesubstance or with the help of spacer groups, so-called homo- orheterobifunctional reagents. Until now, mainly the method of directcoupling has been used which, however, often leads to polymeric productsand not-unequivocally-defined conjugates. Recently, severalchemoimmuno-conjugates (European Patent Application EP 91-117535 911615,European Patent Application EP 90-109268 900516, PCT InternationalPatent Application WO 90-CA251 900809, British (UK) Patent ApplicationGB 83-5104 830224 and European Patent Application EP 89-102370 890210),which were produced using specific bifunctional reagents, were suggestedas cytostatically effective media. Furthermore, from DE 41 22 210 A1,conjugates of tumor-active compounds with transferrin or albumin areknown, wherein the tumor-active compound is activated with N-hydroxysuccinimide and carbodiimide and the thus-obtained mixture is directlycoupled to the carrier protein.

DESCRIPTION OF THE INVENTION

It has now been found that conjugates of transferrin, albumin andpolyethylene glycol, consisting of transferrin, albumin and polyethyleneglycol, with a mass of between 5000 and 200000 Da and, at least, onecytostatic compound derivatized through compounds of maleinimide orN-hydroxysuccinimide, have a tumor-inhibiting effectiveness which isequal or higher than that of the cytostatic compound. Suitable for theproduction of these conjugates of protein or polymer are cytostaticcompounds such as the anthracyclines, doxorubicin, daunorubicin,epirubicin, idarubicin and mitoxandrone, the alkylates, chloroambuciland melphalan, the antimetabolites, methotrexate, 5-fluorouracyl,5′-desoxy-5-fluorouridine and thioguanine, the taxoides, paclitaxel anddocetaxel, the camptothecins, topotecan and 9-aminocamptothecin, thepodophyllotoxin derivatives, etoposide, teniposide and mitopodoside, thevinca alkaloids, vinblastine, vincristine, vindesine and vinorelbine anda compound of the general I, II, III or IV:

n=0-6, X=—NH₂, —OH, —COOH, —O—CO—R—COR*, —NH—CO—R—COR*, wherein R is analiphatic carbon chain with 1-6 carbon atoms or a substituted orunsubstituted phenylene group and R* H, phenyl, alkyl with 1-6 carbon,and the amine functions are provided with a protective group such as thetert.-butyloxycarbonyl protective group, which were derivatized with acompound of maleinimide or N-hydroxysuccinimide. In doing so, thecytostatic compounds are, as a rule, reacted with a maleinimide compoundor N-hydroxysuccinimide compound which has at least one functional groupwhich is suitable for binding to the cytostatic, such as an amino,hydroxy, carbonic acid, carbonic acid chloride, sulfonic acid, sulfonicacid chloride, acid hydracide, or hydrazino, oxycarbonyl chloride,aldehyde or keto group, so that maleinimide derivatives orN-hydroxysuccinimide ester derivatives of cytostatic compounds areprepared, wherein the chemical linkage between the maleinimide compoundand cytostatic compound occurs through an amide, ester, imine,hydrazone, carboxyl hydrazone, oxycarbonyl, acetal or ketal bond. In themaleinimide or N-hydroxysuccinimide compounds which are obtained fromthe compounds of the formulas I-IV, the cytostatic cis-configuredplatinum unit is introduced subsequently, that is, the correspondingplatinum(II)-complexes are obtained, after removal of the protectivegroup, either through reaction with a tetrachloroplatinate salt or withcis-[PtA₂B] (A=halogen, B=(NH₃)₂, ethylene diamine, propane diamine,1,2-diaminocyclohexane).

Through reacting the derivatized cytostatic compounds with native orthiolated transferrin or albumin or with hetero- or homobifunctionalPEGs with a mass of between 5000 and 200 000 Da—Overview 1:

Overview 1: Heterobifunctional PEGs

Hommobifunctional PEGs

conjugates of proteins or polymers are prepared which are producedsimply and effectively, having a high purity, have an excellentwater-solubility in comparison to several of the original cytostaticcompounds, are stable formulations in a physiologic buffer and whichhave an in vitro anti-proliferation effectiveness against human tumorcells which is equal to or better than that of the unbound cytostatics.Furthermore, the conjugates exhibit a equal good or improved anti-tumoreffectiveness in vivo and an improved tolerability. The conjugates ofprotein or polyethylene glycol realized through such couplings, whichare very suitable for a selective treatment of cancer diseases, areobject of the invention and are described in the following.

The method for the synthesis of conjugates of protein or polyethyleneglycol occurs in the conjugates with maleinimide derivatives in foursteps (Steps 1 to 4), in the conjugates with N-hydrosuccinimide esterderivatives in three steps (Steps 1, 2 and 4):

Step 1: Synthesis of maleinimide or N-hydroxysuccinimide compounds

Step 2: Synthesis of maleinimide derivatives or N-hydroxysuccinimideester derivatives of cytostatic compounds

Step 3: Thiolation of the carrier protein

Step 4: Coupling of the cytostatic compound obtained in Step 2 to nativeor thiolated carrier protein or to a PEG shown in Overview 1.

Step 1: Synthesis of Maleinimide or N-hydroxysuccinimide Compounds

The maleinimide compounds are generally produced according to one of thefollowing two methods:

In the first method, maleic acid anhydride is reacted with an aliphaticamino compound H₂N—R—Y, wherein R is an aliphatic C-chain with 1-6carbon atoms or a substituted or unsubstituted benzyl group and Y=—OH,—COOH, —SO₃H, —CH(OC₂H₅)₂, R* —C═O, R*=phenyl or alkyl group with 1 to 6carbon atoms, to yield the corresponding maleaminic acid andsubsequently with triethylamine (Et₅N) in an amount of up to twoequivalents in non-aqueous toluol under azeotropic removal of the waterobtained to yield the corresponding maleinimide compound. The furtherderivatization of the Y group occurs by reacting the —COOH or the —SO₃Hgroup with oxalyl chloride or thionyl chloride to yield thecorresponding acid chlorides, by reacting the hydroxyl group withbis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonylchlorides, by reacting the acetal group —CH(OC₂H₅,)₂ to yield thecorresponding aldehyde with the help of acid-catalytic cleavage such as,for example, through p-toluol sulfonic acid, sulfuric acid or acidicsilica gel, and by reacting acid chloride withN-(tert.-butoxycarbonyl)-alcohol amine orN-(tert.-butoxycarbonyl)-alcohol hydrazine and subsequent cleavage withtrifluoro acetic acid or hydrogen chloride (HCl) in ether,tetrahydrofuran (THF) or dioxan yielding the corresponding amino orhydrazino compounds, respectively.

In the second method, maleic acid anhydride is reacted with an aromaticamino compound H₂N—R—Y, wherein R is a substituted or unsubstitutedphenylene group and Y=—OH, —COOH, —SO₃H, R* —C═O, R*=phenyl or alkylgroup with 1 to 6 carbon atoms, to yield the corresponding maleaminicacid and subsequently with acetic acid anhydride and anhydrous sodiumacetate to yield the corresponding maleinimide compound. The furtherderivatization of the group Y occurs by reacting the —COOH or the —SO₃Hgroup with oxalyl chloride or thionyl chloride to yield thecorresponding acid chlorides, by reacting the hydroxyl group withbis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonylchlorides, by reacting the acid chloride to yield the correspondingaldehydes with the help of LiAl[OC(CH₃)₃]₃H in THF, by reacting the acidchloride in THF or ethyl acetate with t-buytlcarbazate and subsequentcleavage with trifluoroacetic acid or HCl in ether, THF or dioxan toyield the corresponding acid hydrazides and by reacting the acidchlorides with N-(tert.-butoxycarbonyl)-alcohol amine orN-(tert.-butoxycarbonyl)-alcohol hydrazine and subsequent cleavage withtrifluoroacetic acid or HCL in ether, THF or dioxan to yield thecorresponding amino or hydrazino compounds.

The maleinimide compounds of the general formulas VI and VII areproduced by reacting the maleinimide compounds obtained in theabove-mentioned methods, in which Y is —CO—NHNH₂ or —COR* with n=1-6 andR*=H, phenyl, alkyl with 1-6 carbon atoms, with carbonyl compounds ofthe general formula O═CR*—R—Y or with acid hydrazide of the generalformula Y—R—R*CO—NH—NH₂ in anhydrous THF, methanol or ethanol with theoptional addition of toluene-p-sulfonic acid or trifluoroacetic acid. Afurther derivatization of group Y occurs by reacting the COOH— or theSO₃H group with oxalylchloride or with thionyl chloride to yield thecorresponding acid chlorides, by reacting the hydroxy group withbis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonylchlorides.

The bismaleinimide compounds of the general formula XI, wherein twomaleinimide compounds are connected by a group Z, which represents adiaminoalkane, dihydroxyalkane, dihydrazinoalkane or carboxylic aciddihydrazide compound, so that two maleinimide compounds are connectedwith one another via two amide, ester, imine, hydrazone orcarboxylhydrazone bonds, are produced from the above-mentionedmaleinimide compounds, wherein the synthesis of the compounds connectedby the amine bonds occurs by reacting the acid chloride of themaleinimide compounds with diaminoalkane compounds NH₂—(CH₂)_(n)—NH₂,n=2-12, in THF or ethyl acetate with the optional addition of Et₃N, thesynthesis of the compounds connected by an ester bond by reacting theacid chloride of the maleinimide compounds with dihydroxy compoundsHO—(CH₂)_(n)—OH—, n=2-12, in THF or or ethyl acetate with the optionaladdition of Et₃N, the synthesis of the compounds connected by an iminebond by reacting the aldehydes or ketones of the maleinimide compoundswith diaminoalkane compounds NH₂—(CH₂)_(n)—NH₂, n=2-12, in anhydrousTHF, methanol or ethanol with the addition of toluene-p-sulfonic acid ortrifluoroacetic acid and the synthesis of the compounds connected by ahydrazone or carboxyl hydrazone bond occurs by reacting the aldehydes orketones of the maleinimide compounds with dihydrazinoalkane compoundsNH₂—NH—(CH₂)_(n)—NH—NH₂ or carboxylic acid dihydrazines H₂ N—NH—CO—(CH₂)_(n)—CO—NH—NH₂, n=2-12, in anhydrous THF, methanol or ethanol with theaddition of toluene-p-sulfonic acid or trifluoroacetic acid.

The N-hydroxysuccinimide compounds are produced in general by reactingN-hydroxysuccinimide with Y—R—COOH or with Y—R—COCl, wherein R is asubstituted or unsubstituted phenylene group, Y=—OH, —NH₂,—COO—(CH₂)_(n)—OH, —CONH—(CH₂)_(n)NHBOC, —NHNHBOC,—COO—(CH₂)_(n)—NHNHBOC, —SO₃H, —SO₂—NHNHBOC, —CHO, —COR*, —CO—NHNHBOCwith n=1-6 and R*=H, phenyl, alkyl with 1-6 carbon atoms and BOC is thetert.-butyloxycarbonyl protective group, to yield the correspondingN-hydroxysuccinimide ester compound. In so doing, the reaction startingwith Y—R—COOH is performed in an anhydrous solvent, preferablydichloromethane, acetonitril or THF with the addition ofdimethylaminopyridine (DMAP) and a condensation agent, as a rule,N,N′-dicyclohexylcarbodiimide (DCC) orN-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimide metho-p-toluolsulfonate (CMC), to yield the corresponding succinimide esterderivatives. If the acid chloride Y—R—COCl, which is obtained throughchlorination with acid halogenation reagents such as, for example, withoxalylchloride or thionyl chloride, is employed, then the reaction withN-hydroxysuccinimide occurs preferably in anhydrous THF, acetonitril orethyl acetate.

The BOC protective group can subsequently be removed withtrifluoroacetic acid or HCl in ether or dioxan, so that thecorresponding amino or hydrazino compounds as well as the acidhydrazides are obtained as trifluoroacetate or hydrochlorides. A furtherderivatization of group Y occurs by reacting the hydroxy group withbis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonylchlorides. N-Hydroxysuccinimide compounds of the general formulas IX andX are produced by reacting the hydroxysuccinimide compounds obtained inthe above-mentioned methods, in which Y is —CO—NHNH₂ or —COR* with n=1-6and R*=H, phenyl, alkyl with 1-6 carbon atoms, with carbonyl compoundsof the general formula O═CR*—R—Y or with acid hydrazides of the generalformula Y—R—R*CO—NH—NH₂ in anhydrous THF, methanol or ethanol with theoptional addition of toluene-p-sulfonic acid or trifluoroacetic acid.The further derivatization of group Y occurs by reacting the COOH or theSO₃H group with oxalylchloride or with thionyl chloride to yield thecorresponding acid chlorides, by reacting the hydroxy group withbis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonylchlorides.

The isolation of the above-mentioned maleinimide andN-hydroxysuccinimide ester compounds occurs either throughcrystallization, through silica gel column chromotography or throughpreparative HPLC or LPLC on a diol column, as is described in theexamples below.

Step 2: Maleinimide Derivatives or N-hydroxysuccinimide Derivatives ofCytostatic Compounds

Suitable for the reaction with the maleinimide and N-hydroxysuccinimidecompounds obtained in Step 1 are the cytostatic compounds mentioned inclaims 1 to 3. These cytostatic compounds are reacted with themaleinimide and N-hydroxysuccinimide ester compounds described in Step1, so that the maleinimide derivatives and N-hydroxysuccinimide esterderivatives of cytostatic compounds are provided, wherein the chemicallinkage between maleinimide compound or N-hydroxysuccinimide estercompound and cytostatic compound occurs through an amide, ester, imine,hydrazone, carboxyl hydrazone, acetal or ketal bond.

In the case of the anthracyclines, doxorubicin, daunorubicin, epirubicinor idarubicin, the synthesis in detail through the reaction with acidsor acid chlorides, listed in Step 1, of maleinimide orN-hydroxysuccinimide of formulas V to X to yield the correspondinganthracycline-maleinimide derivatives or correspondinganthracycline-hydroxysuccinimide derivatives in a solvent, preferablydimethylformamide (DMF) or THF with the optional addition of a tertiarybase, as a rule, Et₃N, or with the optional addition of DMAP and acondensation agent, as a rule, DCC or CMC, wherein the coupling occursvia the 3′—NH₂ group of the amino sugar of anthracycline as amide anbond,

or through reaction with the aldehydes or ketones of maleinimide orN-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferablyDMF, methanol or ethanol, with the optional addition of an acid, as arule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein thecoupling occurs via the 3′—NH₂ group of the amino sugar of anthracyclineas an imine bond,

or through the reaction with the amines of maleinimide orN-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferablyDMF, methanol or ethanol, with the addition of an acid, as a rule,toluene-p-sulfonic acid or trifluoroacetic acid, wherein the couplingoccurs via the C₁₃-keto position of the anthracycline as an imine bond,

or through the reaction with the acid hydrazides of maleinimide orN-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferablyDMF, methanol or ethanol, with the addition of an acid, as a rule,toluene-p-sulfonic acid or trifluoroacetic acid, wherein the couplingoccurs via the C₁₃-keto position of the anthracycline as a carboxyl orsulfonyl hydrazone bond.

In the case of mitoxandron, the synthesis is performed in detail throughreaction with the acids or acid chlorides of maleinimide orN-hydroxysuccinimide compounds, listed in Step 1, of formulas V to X toyield the corresponding mitoxandron-maleinimide derivatives ormitoxandron-hydroxysuccinimide derivatives in a solvent, preferably DMFor THF with the optional addition of a tertiary base, as a rule, Et₃N,or with the optional addition of DMAP and a condensation agent, as arule, DCC or CMC, wherein the coupling occurs via, at least, one of thealiphatic HO-groups of the mitoxandron as an ester bond,

or through reaction with the aldehydes or ketones of maleinimide orN-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferablyTHF, methanol or ethanol with the optional addition of an acid, as arule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein thecoupling occurs via, at least, one of the aliphatic HO-groups of themitoxandron as an acetal or ketal bond.

In the case of the alkylating agents, chloroambucil and melphalan, thesynthesis is performed in detail through reaction of chloroambucil ormelphalan with the hydroxy compounds of maleinimide orN-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferablyDMF, dichloromethane or THF with the addition of DMAP and a condensationagent, as a rule, DCC or CMC, to yield the corresponding chloroambucilor melphalan-maleinimide derivatives or chloroambucil ormelphalan-hydroxysuccinimide derivatives, respectively, wherein thecoupling occurs via the COOH group of chloroambucil or melphalan as anester bond,

or through reaction of chloroambucil or melphalan, respectively, withacid halogenation reagents such as oxalylchloride or thionyl chloride,to yield the corresponding acid chlorides and subsequent reaction ofacid chlorides in THF or ethyl acetate with t-alkylcarbazates, as arule, tert.-butylcarbazates, or with optional addition of a tertiarybase, as a rule, Et₃N, or through reaction of chloroambucil or melphalanin DMF, THF or ethyl acetate with t-alkylcarbazates, as a rule, DCC orCMC, and subsequent cleavage of the thus-obtained products with acids,as a rule, trifluoroacetic acid or HCl in ether, THF or dioxan, to yieldthe corresponding acid hydrazides of chloroambucil or melphalan,respectively, which, in turn, are reacted with one of the aldehydes orketones, listed in Step 1, of maleinimide or N-hydroxysuccinimidecompounds in a solvent, preferably DMF, methanol or ethanol with theaddition of an acid, as a rule, trifluoroacetic acid ortoluene-p-sulfonic acid, to yield the corresponding maleinimide orN-hydroxysuccinimide carboxyl hydrazone derivatives of chloroambucil ormelphalan, respectively.

In the case of 5-fluorouracil, the synthesis occurs in detail throughreaction with the acid chlorides, listed in Step 1, of the maleinimideor N-hydroxysuccinimide compounds to yield the corresponding maleinimideor N-hydroxysuccinimide derivatives of 5-fluorouracil in a solvent,preferably THF, with the optional addition of a tertiary base, as arule, Et₃N, wherein the coupling occurs via the ¹N- or ³N-position of5-fluorouracil as an acid amide bond,

or through the reaction with the oxycarbonyl chlorides, listed in Step1, of the maleinimide or N-hydroxysuccinimide compounds to yield thecorresponding maleinimide or N-hydroxysuccinimide derivatives of5-fluorouracil in a solvent, preferably THF, with the optional additionof a tertiary base, as a rule, Et₃N, wherein the coupling occurs via the¹N- or ³N-position of 5-fluorouracil as an oxycarbonyl bond,

or through the reaction of 5-fluorouracil with formaldehyde and thecarboxylic acids and sulfonic acids, listed in Step 1, to yield thecorresponding maleinimide or N-hydroxysuccinimide derivatives of5-fluorouracil in a solvent, preferably dichloromethane or THF, with theaddition of DMAP and a condensation agent, as a rule, DCC or CMC,wherein the coupling occurs via the ¹N- or ³N-position of 5-fluorouracilas a carbamoyloxymethyl bond.

In the case of 5′-desoxy-5-fluorouridine, the synthesis is performed indetail through reaction with the acid chlorides, listed in Step 1, ofthe maleinimide or N-hydroxysuccinimide compounds of formulas V to X toyield the corresponding maleinimide or N-hydroxysuccinimide derivativesof 5′-desoxy-5-fluorouridine in a solvent, preferably THF, with theoptional addition of a tertiary base, as a rule, Et₃N, wherein thecoupling occurs via the 2′—HO or 3′—HO group of5′-desoxy-5-fluorouridine as an ester bond,

or through the reaction with the aldehydes or ketones, listed in Step 1,of the maleinimide or N-hydroxysuccinimide compounds in a solvent,preferably THF, methanol or ethanol, with the addition of an acid, as arule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein thecoupling occurs via the 2′—HO and/or 3′—HO group of5′-desoxy-5-fluorouridine as an acetal or ketal bond.

In the case of thioguanine, the synthesis is performed in detail throughthe reaction with the acid chlorides, listed in Step 1, of themaleinimide or N-hydroxysuccinimide compounds of formulas V to X toyield the corresponding maleinimide or N-hydroxysuccinimide derivativesof thioguanine in a solvent, preferably DMF, with the optional additionof a tertiary base, as a rule, Et₃N, wherein the coupling occurs via theH₂N group of thioguanine as an amide bond,

or through the reaction with the aldehydes or ketones, listed in Step 1,of the maleinimide or N-hydroxysuccinimide compounds in a solvent,preferably DMF, methanol or ethanol, with the addition of an acid, as arule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein thecoupling occurs via the H₂N group of thioguanine as an imine bond.

In the case of methotrexate, the synthesis is performed in detailthrough the reaction of methotrexate with the hydroxy or amino compoundsof maleinimide or N-hydroxysuccinimide compounds, listed in Step 1, in asolvent, preferably DMF or dimethylsulfoxide with the addition of DMAPand a condensation agent, as a rule, DCC or CMC, to yield thecorresponding methotrexate-maleinimide derivatives ormethotrexate-hydroxysuccinimide derivatives, respectively, wherein thecoupling occurs either via the α-COOH group or γ-COOH group or via bothCOOH groups of methotrexate as an ester or amide bond,

or through the reaction of methotrexate with alkylcarbazates, as a rule,t-butylcarbazate, in a solvent, preferably DMF or dimethyl sulfoxidewith the addition of DMAP and a condensation agent, as a rule, DCC orCMC, and subsequent cleavage with acids, as a rule, trifluoroacetic acidor HCl in ether, THF or dioxan, to yield the corresponding acidhydrazides of methotrexate, wherein the acid hydrazide group wasintroduced at either the α-COOH group or γ-COOH group or at both COOHgroups of methotrexate, and the thus-obtained acid hydrazide derivativesof methotrexate are reacted now with one of the aldehydes or ketones,listed is Step 1, of the N-hydroxysuccinimide compounds in a solvent,preferably DMF, methanol, THF or ethanol, with the addition of an acid,as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, to yield thecorresponding N-hydroxysuccinimide carboxylhydrazone derivatives ofmethotrexate.

In the case of the taxoides, paclitaxel and docetaxel, the synthesis isperformed in detail through the reaction with the acids or acidchlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimidecompounds of formulas V to X to yield the correspondingtaxoid-maleinimide derivatives or taxoid-hydroxysuccinimide derivativesin a solvent, preferably DMF or THF with the optional addition of atertiary base, Et₃N, or with the optional addition of DMAP and acondensation agent, as a rule, DCC or CMC, wherein the coupling occursvia the C₇— or C₁₀—OH group of the taxoid as an ester bond,

or through the reaction with the amines or hydrazines, listed in Step 1,of the maleinimide or N-hydroxysuccinimide compounds in a solvent,preferably DMF, methanol or ethanol, with the addition of an acid, as arule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein thecoupling occurs via the C₉-keto position of the taxoid as an imine orhydrazone bond,

or through the reaction with the acid hydrazides, listed in Step 1, ofthe maleinimide or N-hydroxysuccinimide compounds in a solution,preferably DMF, methanol or ethanol, with the addition of an acid, as arule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein thecoupling occurs via the C₉-keto position of the taxoid as a carboxyl orsulfonyl hydrazone bond.

In the case of the camptothecines, topotecan or 9-aminocamptothecine,the synthesis is performed in detail through the reaction with the acidsor acid chlorides, listed in Step 1, of the maleinimide orN-hydroxysuccinimide compounds of formulas V to X to yield thecorresponding taxoid maleinimide derivatives ortaxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF orTHF with the optional addition of a tertiary base, Et₃N, or with theoptional addition of DMAP and a condensation agent, as a rule, DCC orCMC, wherein the coupling occurs via the C₁₀—OH group of the topotecanas an ester bond or via the C₉—NH₂ group of the 9-aminocamptothecin asan amide bond,

or through the reaction of 9-aminocamptothecin with the aldehydes orketones, listed in Step 1, of of the maleinimide or N-hydroxysuccinimidecompounds in a solution, preferably DMF, methanol or ethanol, with theoptional addition of an acid, as a rule, toluene-p-sulfonic acid ortrifluoroacetic acid, wherein the coupling occurs via the C₉—NH₂ groupas an imine bond.

In the case of the podophyllotoxin derivatives, etoposide, teniposideand mitopodozide, the synthesis is performed in detail through reactionwith the acids or acid chlorides, listed in Step 1, of maleinimide orN-hydroxysuccinimide compounds of formulas V to X to yield thecorresponding taxoid-maleinimide derivatives ortaxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF,dichloromethane or THF with the optional addition of a tertiary base, asa rule, Et₃N, or with the optional addition of DMAP and a condensationagent, as a rule, DCC or CMC, wherein the coupling occurs via one of thealiphatic HO-groups of the podophyllotoxin derivative as an ester bond.

In the case of the vinca alkaloids, vinblastine, vincristine, vindesineand vinorelbine, the synthesis occurs in detail through the reactionwith the acids or acid chlorides, listed in Step 1, of maleinimide orN-hydroxysuccinimide compounds of formulas V to X to yield thecorresponding taxoid-maleinimide derivatives ortaxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF,dichloromethane or THF with the optional addition of a tertiary base, asa rule, Et₃N, or with the optional addition of DMAP and a condensationagent, as a rule, DCC or CMC, wherein the coupling occurs via one of thealiphatic HO-groups of the vinca alkaloid as an ester bond.

In the case of maleinimide or N-hydroxysuccinimide derivatives of thecis-configured platinum(II)-complexes, the synthesis occurs in detailthrough the reaction of the corresponding amino compoundsH₂N—CH₂CH₂—NH—(CH₂)_(n)—X, (H₂N—CH₂)₂CH—(CH₂)_(n)—X orH₂N—CH₂CH(NH₂)—(CH₂)_(n)—X (general formulas I, II and III), wherein oneor two of the primary or secondary amino groups has been protected witha BOC group (reaction with bis-tert.-butyloxy carbonyl anhydride) and Xis —NH₂ or —OH, with the acids or acid chlorides, listed in Step 1, ofmaleinimide or N-hydroxysuccinimide compounds of the general formulasV-X in a solvent, preferably THF or ethyl acetate, with the optionaladdition of a tertiary base, as a rule, Et₃N, or with the optionaladdition of DMAP and a condensation agent, as a rule, DCC or CMC, toyield the corresponding BOC-protected maleinimide or hydroxysuccinimidederivatives which then are converted by means of trifluoroacetic acid orHCl in ether, THF or dioxan through cleavage-off of the BOC group intothe corresponding trifluoroacetate or hydrochloride and finally throughreaction with a tetrachloro-platinate(II) salt, preferably potassiumtetrachloro-platinate(II), in water, salt buffers, DMF, DMF/watermixtures, THF/water mixtures or DMF/methanol mixtures, into thecorresponding platinum(II)-complexes, wherein the coupling occurs viathe terminal HO group as an ester bond or via the terminal H₂N group asan acid amide bond, or through the reaction of the corresponding aminocompounds H₂N—CH₂CH₂—NH—(CH₂)_(n)—X, (H₂N—CH₂)₂CH—(CH₂)_(n)—X orH₂N—CH₂CH(NH₂)—(CH₂)_(n)—X (general formulas I, II and III), wherein oneor two of the primary or secondary amino groups has been protected witha BOC group (reaction with bis-tert.-butyloxy carbonyl anhydride) and Xis —NH₂ or —OH, with compounds of the type HOOC—R—COCR* or ClOC—R—COCR*(R is an aliphatic carbon chain with 1-6 carbon atoms or a substitutedor unsubstituted phenylene group, and R* is H, phenyl, alkyl with 1-6carbon atoms) in a solvent, preferably THF or ethyl acetate, with theoptional addition of a tertiary base, as a rule, Et₃N, or with theoptional addition of DMAP and a condensation agent, as a rule, DCC orCMC, to yield the corresponding BOC-protected maleinimide orhydroxysuccinimide derivatives which now have a further carbonylfunction which, in the following, are reacted with the amines, acidhydrazides or hydrazines, listed in Step 1, of the maleinimide orN-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol orethanol, with the addition of acid, as a rule, toluene-p-sulfonic acidor trifluoroacetic acid, to yield the corresponding imine,carboxylhydrazone or hydrazone derivatives, which then again areconverted by means of trifluoroacetic acid or HCl in ether, THF ordioxan through cleavage-off of the BOC group into the correspondingtrifluoroacetate or hydrochloride and finally through reaction with atetrachloro-platinate(II) salt, preferably potassiumtetrachloro-platinate(II), in water, salt buffers, DMF, DMF/watermixtures, THF/water mixtures or DMF/methanol mixtures, into thecorresponding platinum(II)-complexes, or through the reaction of thecorresponding amino compounds H₂N—CH₂CH₂—NH—(CH₂)_(n)—X,(H₂N—CH₂)₂CH—(CH₂)_(n)—X or H₂N—CH₂CH(NH₂)—(CH₂)_(n)—X (general formulasI, II and III), wherein one or two of the primary or secondary aminogroups has been protected with a BOC group (reaction withbis-tert.-butyloxy carbonyl anhydride) and X is COOH or this carbonylgroup was converted using acid halogenation reagents such as thionylchloride or oxalyl chloride into the acid chloride, with compounds ofthe type HOR—COCR* or H₂N—R—COCR* (R is an aliphatic carbon chain with1-6 carbon atoms or a substituted or unsubstituted phenylene group, andR* is H, phenyl, alkyl with 1-6 carbon atoms) in a solvent, preferablyTHF or ethyl acetate, with the optional addition of a tertiary base, asa rule, Et₃N, or with the optional addition of DMAP and a condensationagent, as a rule, DCC or CMC, to yield the corresponding BOC-protectedmaleinimide or hydroxysuccinimide derivatives which now have a furthercarbonyl function which, in the following, are reacted with the amines,acid hydrazides or hydrazines, listed in Step 1, of the maleinimide orN-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol orethanol, with the addition of acid, as a rule, toluene-p-sulfonic acidor trifluoroacetic acid, to yield the corresponding imine,carboxylhydrazone or hydrazone derivatives, which then again areconverted by means of trifluoroacetic acid or HCl in ether, THF ordioxan through cleavage-off of the BOC group into the correspondingtrifluoroacetate or hydrochloride and finally through reaction with atetrachloro-platinate(II) salt, preferably potassiumtetrachloro-platinate(II), in water, salt buffers, DMF, DMF/watermixtures, THF/water mixtures or DMF/methanol mixtures, into thecorresponding platinum(II)-complexes.

In the case of maleinimide or N-hydroxysuccinimide derivatives withmalonic acid derivatives of the general formula IV(HOOC)₂—CH—(CH₂)_(n)—X to the cis-configured platinum(II)-complexes, thesynthesis occurs analogous to the above-described complexes, wherein theplatinum(II)-complexes are obtained by reacting the maleinimide orN-hydroxysuccinimide derivatives with malonic acid derivatives withcis-[PtA₂B] (A=halogen, preferably Cl or J, B=(NH₃)₂, ethylene diamine,propane diamine, 1,2-diaminocyclohexane) to yield the correspondingplatinum(II)-complexes in a solvent, such as water salt buffers, DMF,DMF/water mixtures, THF/water mixtures or DMF/methanol mixtures with theaddition of a hydroxide solution, preferably aqueous KOH. The reactioncan optionally be carried out in the presence of silver nitrate (AgNO₃)or silver sulfate (Ag₂SO₄). The platinum complex is obtained throughcrystallization or through addition of a solvent, preferablydiethylether or THF.

The isolation of the above-mentioned maleinimide or N-hydroxysuccinimidecytostatic compounds, respectively, occurs either throughcrystallization, through silica gel column chromatography or throughpreparative HPLC or LPLC on a reverse-phase (C8 or C18) or diol column,as is described in the examples below.

Step 3: Thiolation of the Carrier Protein

Sulfohydryl groups (HS groups) are introduced through reaction of thecarrier protein with a thiolation reagent, preferably iminothiolan, intohuman serum transferrin and serum albumin. The thiolation occurs in asalt buffer, as a rule, in 0.1 M sodium borate, 0.15 M NaCl, 0.001 MEDTA—pH=8.0, with an excess of thiolation reagent (2- to 100-foldexcess) and subsequent gel filtration (for example, Sephadex® G10 ofG25) with a salt buffer such as 0.025 M sodium borate, 0.15 M NaCl—pH6.0-7.5 or 0.004 M phosphate, 0.15 M NaCl—pH 6.0-7.5. The concentrationof protein after completed gel filtration is determined through theextinction coefficient at 280 nm and is, as a rule, in the range ofbetween 1.0×10⁻⁴ and 5.0×10⁻³ M. The number of the introduced HS groupsis determined with Ellmann's reagent at 412 nm. Through variation of thereaction conditions, 1 to 30 HS groups can be introduced on the average.The thiolated transferrin or albumin solution is employed directly forthe synthesis of the conjugates.

Step 4: Coupling of the Cytostatic Maleinimide or N-hydroxysuccinimideCompounds to the Native or Thiolated Carrier Protein or to aPolyethylene Glycol Shown in Overview 1

For the coupling of the cytostatic maleinimide or N-hydroxysuccinimidecompounds to PEGs, PEGs are employed which have one or two HO—, HS— orH₂N groups and a mass of between 5,000 and 200,000 Da, preferablybetween 20,000 and 70,000 Da. Corresponding compounds are notcommercially available. In the following, polyethylene glycols havingone or two HS groups are shortened with HS-PEG or HS-PEG-SH, and thePEGs having one or two H₂N groups are shortened with H₂N-PEG orH₂N-PEG-NH₂.

Coupling of the cytostatic maleinimide derivatives to the thiolatedcarrier protein or to HS-PEG, HS-PEG-SH, H₂N-PEG or H₂N-PEG-NH₂: Thecytostatic maleinimide derivatives (see Step 2) are reacted withthiolated transferrin, albumin (see Step 3), HS-PEG, HS-PEG-SH, H₂N-PEGor H₂N-PEG-NH₂ at room temperature. In so doing, to the thiolatedprotein, HS-PEG, HS-PEG-SH, H₂N-PEG or H₂N-PEG-NH₂, which is present ina degassed salt buffer such as 0.025 M sodium borate, 0.15 M NaCl—pH 6.0to 7.5 or 0.004 M phosphate, 0.15 M NaCl—pH 6.0 to 7.5, an approximately1.1- to 10-fold excess of the cytostatic maleinimide derivative is added(in terms of the number of available HS groups in the protein or PEG),dissolved in a minimal amount of solvent, as a rule, DMF,dimethylsulfoxide, water, ethanol, methanol, acetonitril or THF(approximately 1 to 10% of the volume of the thiolated sample). Afterapproximately 5 to 120 minutes, the solution is centrifuged, and theformed protein conjugate or PEG conjugate is separated off throughsubsequent gel filtration (for example, Sephadex® G10, G25 or LH20) in adegassed salt buffer such as 0.025 M sodium borate, 0.15 M NaCl—pH6.0-7.5, 0.004 M phosphate, 0.15 M NaCl—pH 6.0-7.5 or 0.1-0.2 M NaHCO₃,or in methanol or THF, from the excess cytostatic maleinimidederivative. It can be advantageous to dilute the thiolated proteinsolution prior to the addition of the maleinimide derivative with a saltbuffer and to add the maleinimide derivative, which is dissolved in aminimal amount of solvent, and subsequently to concentrate the solutionafter 5-20 minutes with a customary commercial concentrator and toisolate the protein conjugate, as described above. Further, the solutionof the thus-obtained protein conjugate or of the PEG conjugate can beconcentrated with a customary commercial concentrator or the solventremoved under a high vacuum.

Coupling of the cytostatic N-hydroxysuccinimide derivatives to thenative carrier protein or to HO-PEG, HO-PEG-OH, H₂N-PEG or H₂N-PEG-NH₂:The cytostatic N-hydroxysuccinimide derivatives (see Step 2) are reactedwith transferrin, albumin, HO-PEG, HO-PEG-OH, H₂N-PEG or H₂N-PEG-NH₂ atroom temperature. In so doing, to the protein, HO-PEG, HO-PEG-OH,H₂N-PEG or H₂N-PEG-NH₂, which is located in a degassed salt buffer suchas 0.025 M sodium borate, 0.15 M NACl—pH 6.0 to 8.0 or 0.004 Mphosphate, 0.15 M NaCl—pH 6.0 to 8.0, an approximately 1.1- to 50-foldexcess of the cytostatic N-hydroxysuccinimide derivative is added,dissolved in a minimal amount of solvent, as a rule, DMF,dimethylsulfoxide, water, ethanol, methanol, acetonitril or THF(approximately 1 to 10% of the volume of the thiolated sample). Afterapproximately 5 minutes to 48 hours, the solution is centrifuged, andthe formed protein conjugate or PEG conjugate is separated off throughsubsequent gel filtration (for example, Sephadex® G10, G25 or LH20) in adegassed salt buffer such as 0.025 M sodium borate, 0.15 M NaCl—pH6.0-7.5, 0.004 M phosphate, 0.15 M NaCl—pH 6.0-7.5 or 0.1-0.2 M NaHCO₃,or in methanol or THF, from the excess cytostatic N-hydroxysuccinimidederivative. The solution of the thus-obtained protein conjugate or ofthe PEG conjugate can be concentrated with a customary commercialconcentrator or the solvent removed under a high vacuum.

The number of the cytostatic maleinimide derivatives orN-hydroxysuccinimide derivatives bound to the carrier protein or to thepolyethylene glycol is specified either through a photometricconcentration determination at the absorbed wavelength of the cytostaticmaleinimide or N-hydroxysuccinimide derivative (typically between 220and 600 nm) and/or through colorimetric determination which, in the caseof the conjugates of chloroambucil and melphalan, is performed with theaid of the NBP test (Epstein, J., Rosenthal, R. W., Ess, R. J. Anal.Chem. (1955), 27, 1435-1439), in the case of the conjugates of5-fluorouracil and 5′-desoxy-5-fluorouridine with the aid of an assayaccording to Habib (Habib, S. T., Talanta (1981), 28, 685-87) and in thecase of the conjugates of the cis-configured platinum(II)-analogues withthe aid of a determination according to Gonias and Pizzo (Gonias, S. L.,Pizzo, S. V. J.Biol.Chem. (1982), 258, 5764-5769) or according to atomicabsorption spectroscopy (AAS).

On the average, through the above-described way, approximately 1-30molecules of the cytostatic compound is bound to one molecule of proteinor 1-2 molecules of the cytostatic compound to one molecule of the PEGs.The purity of the protein conjugate or the PEG conjugate is checkedthrough HPLC with the aid of an analytical column (Bio-Sil SEC 250, (300mm×7.8 mm) from Bio-RAD, mobile phase: as a rule, 0.15 M NaCl, 0.01 MNaH₂PO₄, 5% of CH₃CN—pH 7.0 or Nucleogel® aqua-OH 40 or 60, fromMacherey and Nagel, mobile phase: as a rule, 0.1 M NaCl, 0.004 MNaH₂PO₄, 30% methanol—pH 7.0). In so doing, the transferrin, albumin andpolyethylene glycol conjugates exhibit a purity of >90%.

The protein conjugates or PEG conjugates can be stored in a dissolvedform at 0-5° C., in frozen form at T=−20° C. or −78° C. Furthermore, itis possible to lyophilize the solution of the conjugate and store thelyophilisate at +5 to −78° C.

Object of the invention are also such chemoimmuno-conjugates consistingof albumin which, according to one of the claims 1 to 3, is loaded withapproximately two to thirty equivalents of a cytostatic compound and isconjugated, with a protein, for example, with transferrin or with amonoclonal antibody which is directed against a tumor-associatedantigen, preferably, however, with transferrin, via one of thebismaleinimide compounds mentioned in claim 3 or via an aliphatic oraromatic bismaleinimide compound. In so doing, approximately 80-90% ofthe thiol groups introduced into albumin with the cytostatic maleinimidederivative, dissolved in a minimal amount of solvent, as a rule, DMF,dimethylsulfoxide, ethanol, methanol, acetonitril or THF (approximately1-10% of the volume of the thiolated sample) are reacted and, afterapproximately 5 to 60 minutes, a 1.5- to 20-fold excess of thebismaleinimide compound, dissolved in a minimal amount of solvent, as arule, DMF, dimethylsulfoxide, ethanol, methanol, acetonitril or THF(approximately 1-10% of the volume of the thiolated sample) is added.After approximately 5 to 20 minutes, the solution is centrifuged, andthe formed protein conjugate separated off through subsequent gelfiltration (for example, Sephadex® G10 or G25) in a salt buffer, as arule, 0.025 M sodium borate, 0.15 M NaCl—pH 6.0-7.5 or 0.004 Mphosphate, 0.15 M NaCl—pH 6.0-7.5, from the cytostatic maleinimidederivative and the bismaleinimide compound. Then the thus-modifiedalbumin conjugate is reacted with one of the proteins mentioned abovewhich contains approximately 1.5 thiol groups on the average, and theresulting chemoimmuno-conjugate which now consists of an albuminmolecule loaded with a cytostatic compound and of the above-mentionedprotein, is isolated with the aid of a Superdex-200 column (company:Pharmacia) or a hydroxyl apetite column (company: Pharmacia or Bio-Rad)in a salt buffer, as a rule, 0.025 M sodium borate, 0.15 M NaCl—pH6.5-8.0, 0.004 M phosphate, 0.15 M NaCl—pH 6.5-8.0.

It is also possible to, first, react one of the above-mentionedproteins, which contains 1.5 thiol groups on the average, with a 1.5- to10-fold excess of the bismaleinimide compound and then to separate theformed protein conjugate through subsequent gel filtration (for example,Sephadex® G10 or G25) in a degassed salt buffer such as 0.025 M sodiumborate, 0.15 M NaCl—pH 6.0-7.5, 0.004 M phosphate, 0.15 M NaCl—pH6.0-7.5 or 0.1-0.2 M NaHCO₃, from the bismaleinimide compound, and thento react the thus-modified protein conjugate, which now contains 1.5equivalents of the bismaleinimide compound on the average, with themodified albumin conjugate, wherein approximately 80-90% of the thiolgroups introduced into the albumin have already been reacted with acytostatic maleinimide derivative. The resulting chemoimmuno-conjugatewhich now consists of an albumin molecule loaded with a cytostaticcompound and of the above-mentioned protein, is isolated with the aid ofa Superdex-200 column (company: Pharmacia) or a hydroxyl apatite column(company: Pharmacia or Bio-Rad) in a degassed salt buffer, such as,0.025 M sodium borate, 0.15 M NaCl—pH 6.0-8.0, 0.004 M phosphate, 0.15 MNaCl—pH 6.0-8.0 or 0.1-0.2 M NaHCO₃.

The following examples describe the invention in more detail withoutlimiting it.

EMBODIMENT EXAMPLES Step 1: Synthesis of Maleinimide- andHydroxysuccinimide Compounds p-Maleinimidobenzophenone

14.0 g (70 mmol) of p-aminobenzophenone were dissolved in 70 ml ofacetone and 7.0 g (70 mmol) maleic acid anhydride, dissolved in 40 ml ofacetone, were added drop-wise over a period of 15 minutes at roomtemperature. This set-up was stirred for 3 h at room temperature. Thenthe precipitation was filtered off, washed with ether and dried under avacuum (yield: 93%). 19.3 g (65.4 mmol) of N-p-benzophenyl)-maleic acidamide and 2.6 g (31.0 mmol) of anhydrous sodium acetate were dissolvedat 55° C. in 50 ml of acetic acid anhydride and stirred at thistemperature for 2 h. Subsequently, the acetic acid anhydride was removedat 60° C. under a water jet vacuum. To the residue, 300 ml of water wasadded and stirred for 2 h at 70° C. The precipitation precipitatingduring this time was suctioned off, washed with water, and dissolved andrecrystallized out of acetone (yield: 94%); melting point: 150° C.;R_(f)-value: 0.70 (acetic ester/hexane 6/1); calculated: C: 73.64% H:3.97% N: 5.05%, C₁₇H₁₁NO₃; found: C: 73.15% H: 3.96% N: 5.00%.

p-Maleinimidophenylacetic acid

25.0 g (166.9 mmol) of p-aminophenylacetic acid were suspended in 250 mlof methanol and while heating brought into the solution with theaddition of 500 ml. 19.25 g (166.9 mmol) of maleic acid anhydride weredissolved in 100 ml of acetone and, over a period of 60 minutes, addeddrop-wise to the solution previously cooled to 40° C. The set-up wasstirred for 60 minutes at room temperature. Then the reaction mixturewas concentrated to approximately 300 ml and set aside to cool at −20°C. The precipitation was suctioned off, washed with acetone and driedunder a high vacuum (yield: 82%). 18.0 g (72 mmol) of N-(4-acetic acidphenyl) maleic acid amide were suspended in 2.4 liters of toluene andheated to reflux. While heating, 14.7 g (20.2 ml, 144 mmol) of triethylamine were added and subsequently heated for 2 hours to reflux at thewater separator. Then it was destined off from the formed red oil andthe solvent was removed under a vacuum. The yellow residue was dissolvedin 300 ml of water and set to pH=2 with 1 M HCl. The acidic solution wasextracted with acetic ester (6×50 ml). The combined acetic ester phaseswere dried via sodium sulfate and the solvent removed under a vacuum.The product was recrystallized from acetic ester/hexane (yield: 51%yellow crystals); melting point: 158° C.; R_(f)-value: 0.45 (aceticester/methanol 2/1); calculated: C: 62.34% H: 3.92% N: 6.09%, C₁₂H₉NO₄;found: C: 61.84% H: 4.43% N: 5.59%.

p-Maleinimidophenylacetic acid chloride

1.0 g (4.33 mmol) of p-maleinimidophenylacetic acid were suspended in 25ml of dichloromethane and diluted with a 2.5-fold excess of oxalic aciddichloride (1.37 g, 945 μl; 10.82 mmol). The reaction mixture was heatedto 30-40° C. with the exclusion of moisture and stirred for 15 h. Thenthe solvent was removed under a vacuum and dried under a high vacuum.Crystallization from toluene yielded a yellow powder (yield: 59%);melting point: 154° C.; R_(f)-value: 0.29 (acetic ester/hexane 4/1)

Elementary analysis: calculated: C: 58.91% H: 3.30% N: 5.75%, Cl: 14.49%(C₁₂H₈NO₃Cl) found: C: 60.61% H: 3.64% N: 5.13% Cl: 13.80%

p-Maleinimidophenylacetic acid hydrazide. CF₃COOH

3.5 g (14 mmol) of p-maleinimidophenylacetic acid chloride weredissolved together with tert.-butylcarbazate (2.87 g, 21.7 mmol) in 50ml of anhydrous tetrahydrofuran and stirred for 2 h at room temperature.The tetrahydrofuran was removed under a high vacuum and the residuetaken up in 500 ml of acetic ester. The acetic ester phase was shakenout twice with 125 ml of water each and then dried via anhydrous sodiumsulfate. The solution was concentrated by evaporation, and dissolved andrecrystallized from acetic ester/hexane. The product was suctioned offand dried under a high vacuum (yield: 90%); melting point: decompositionat 152° C.; R_(f)-value: 0.50 (tetrahydrofuran/hexane 3/1). 2.5 g (7.25mmol) p-maleinimidophenylacetic acid hydrazino-tert.-butylcarbazate weredissolved in 12 ml of trifluoroacetic acid and stirred for 1 h at roomtemperature. The trifluoroacetic acid was subsequently removed under ahigh vacuum and the residue suspended in 50 ml of ether. The precipitatewas suctioned off, washed with dry ether and dried under a high vacuum(yield: 82% yellowish powder); melting point: 112° C.; R_(f)-value: 0.06(tetrahydrofuran/hexane 3/1)

(C₁₄H₁₂N₃O₅F₃) calculated: C: 46.80% H: 3.34% N: 11.70% found: C: 46.95%H: 3.24% N: 11.51%

Maleinimidoacetaldehyde

91.6 g (100 ml, 689 mmol) aminoacetaldehyde diethylacetal were dissolvedin 200 ml of acetic ester. Thereafter, 67.5 g (689 mmol) of maleic acidanhydride, dissolved in 200 ml of acetic acid, were added drop-wisewhile stirring and cooling with ice within 60 minutes. Stirring wasperformed for 1 h at room temperature. Then the reaction mixture wasconcentrated to the half and set to cool at −20° C. After 24 h, theobtained precipitate was suctioned off under a vacuum, washed withacetic ester and ether, and subsequently dried under a high vacuum.(Yield: 90%). 30.95 g (133.8 mmol) of N-(acetaldehydediethylacetal)maleic acid amide were dissolved in 900 ml of toluene,14.2 g (19.6 ml, 140.5 mmol) of triethylamine were added thereto and thereaction mixture boiled for 15 h at the water separator. Thereafter, thesolvent was removed under a vacuum. The remaining syrup was taken up in350 ml of diethyl ether and extracted 3× with 50 ml of water each. Theether phase was dried over Na₂SO₄, the solvent removed under a vacuumand the residue purified using column chromatography (aceticester/hexane 1/1; yield: 36% of colorless syrup); ; R_(f)-value: 0.49(acetic ester/hexane 1/1)

(C₁₀H₁₅NO₄) calculated: C: 58.15% H: 7.49% N: 6.17% found: C: 58.10% H:6.98% N: 6.35%

20.1 g (94.3 mmol) of maleinimidoacetaldehyde diethylacetal weredissolved in 350 ml of dichloromethane and 40.2 of silica gel 60 wereadded while stirring. 4.0 g of 30-% sulfuric acid were added andrefluxed for 60 h. Then the silica gel was filtered off and the reactionsolution was extracted with 4×50 ml of water. The dichloromethane wasdried over sodium sulfate and concentrated through evaporation. Theresidual syrup was dissolved and recrystallized from acetic ester/hexane(1:10) (yield: 15% white crystal); melting point: 68-69° C.;R_(f)-value: 0.52 (THF/hexane 3/1)

(C₆H₅NO₃) calculated: C: 51.76% H: 3.62% N: 10.06% found: C: 51.48% H:4.14% N:  9.60%

2-Maleinimidoethyl chloroformate

2.0 g (14.2 mmol) 2-hydroxyethylmaleinimide were dissolved in 150 ml ofabsolute dichloromethane and diluted with 1.56 g (4.8 mmol) oftriphosgene. After the addition of 479 mg (660 μl; 4.8 mmol) oftriethylamine, the set-up was stirred for 72 h at room temperature,subsequently the solvent was removed under a vacuum and the residue waspurified using column chromatography via silica gel (running medium:acetic ester/hexane 2/1; yield: 80% colorless solid); melting point: 45°C.; R_(f)-value: 0.69 (acetic ester/hexane 2/1); calculated: C: 41.25%H: 2.95% N: 6.88 %, Cl: 17.41% (C₇H₆NO₄Cl) found: C: 41.11% H: 3.00% N:6.88% Cl: 16.94%

Synthesis of Bismaleinimide Compounds:

1,4-diamino-N,N′-di-m-maleinimidobenzyl-butane

2.36 g (10 mmol) of maleinimidobenzoic acid chloride as well as 0.44 g(5 mmol) of 1,4-diaminobutane were dissolved each in 60 ml of ethylacetate. In a three-necked flask, 40 ml of ethyl acetate were placed.While stirring at room temperature, both of the solutions were addeddrop-wise to synchronously over a period of 30 minutes. During thedrop-wise addition, a light-yellow precipitate precipitated out. Theset-up was stirred for 2 h, the precipitate suctioned off and washedwith diethylether. The light-yellow solid was dissolved andrecrystallized out from acetone, washed with water and then again withether and dried under a vacuum. 2.1 g (3.9 mmol, 75% of the theory) ofthe product were obtained as a yellow-white solid, DC: silica gel,THF/hexane 4:1, R_(f)-value: 0.30 (THF/hexane 3/1), Fp.: 221° C.(decomposition); C₂₆H₂₂N₄O₆(486 g/mol) calculated: C: 64.20% H: 4.53% N:11.52% O: 19.75%; found: C: 62.57% H: 4.55% N: 10.67%

Diacylhydrazone from adipic acid dihydrazide andm-maleinimidoacetophenone

1.0 g (4.65 mmol) m-maleinimidoacetophenone was dissolved together with368 mg (2.11 mmol) of adipic acid dihydrazide in 40 ml of absolutemethanol. To the reaction mixture was added 100 μl of trifluoroaceticacid and stirred for 15 h at room temperature. The precipitate thusprecipitating out was suctioned off and washed 2× with 30 ml of methanoleach as well as 4× with 50 ml of diethylether each. Then the product wasdried under a vacuum; DC: silica gel, THF/hexane 4:1, R_(f)=: 0.36, Fp.:240° C. (decomposition); C₃₀H₂₈N₆O₆ (568 g/mol) calculated: C: 63.38% H:4.93% N: 14.79%; found: C: 63.18% H: 4.83% N: 15.03%

Synthesis of N-hydroxysuccinimide Ester Compounds:

4-acetophenone carboxylic acid-(N-hydroxysuccinimide)-ester

2.2 g (1.2 mmol) acetophenone-4-carboxylic acid and 1.52 g (1.3 mmol)and 20 mg of DMAP are dissolved in 40 ml of tetrahydrofuran and then 2.7g of DCC, dissolved in 20 ml of THF, were added drop-wise while coolingwithin 1 h. The reaction mixture is stirred while cooling over a periodof 12 h, filtered, THF removed under a vacuum, and the residue dissolvedand recrystallized from acetic ester/methanol 1:1, R_(f)=0.71, Fp.: 140°C.; C₁₃H₁₁NO₅ (261 g/mol); calculated: C: 59.77% H: 4.21% N: 5.36%;found: C: 60.1% H: 4.2% N: 5.0%

4-Carboxylbenzoylhydrazide

25 g (138.9 mmol) of terephthalic acid monomethylester are suspended in200 ml of THF and heated to reflux. In the portion-wise addition of 40.5ml (833.4 mmol, 6 eq) of hydrazine monohydrate, the product precipitatesout as a white solid. The excess THF is distilled off and the residuedissolved in water. Through the addition of conc. HCl, a pH ofapproximately 4 is set. A white precipitate is formed which is suctionedoff and washed with half-conc. HCl. The product is dried under a vacuum,and dissolved and recrystallized from methanolic caustic soda solution.Obtained are 24.84 g (138 mmol) of the product in the form of colorlesscrystal needles, corresponding to 99.3% of the theoretically possibleyield. C₈H₈N₂O₃ (180 g/mol); calculated: C: 53.33% H: 4.44% N: 15.55%;found: C: 52.41% H: 4.26% N: 14.68%

N-Tertiarybutyloxycarbonyl-4-carboxybenzoylhydrazide

20 g (0.11 mol) 4-carboxybenzoylhydrazide are suspended in 200 ml of THFand diluted with a solution of 23.98 g (0.11 mol) ofbis-tert.-butyldicarbonate in 50 ml of THF. After 48 h of stirring atroom temperature, the THF is removed under a vacuum, the oily residuetaken up in 400 ml of ethylacetate and extracted 4 times each with 100ml of water. After drying of the organic phase over Na₂SO₄, it isconcentrated to half and cooled to 4° C. Colorless crystals are formedwhich are suctioned off and washed with diethylether. Obtained are 30.3g (0.108 mmol) of the product, corresponding to 98.4% of thetheoretically possible yield.

C₁₃H₁₆N₂O₅ (280 g/mol) calculated: C: 55.71% H: 5.71% N: 10.00% found:C: 56.94% H: 5.56% N:  9.79%

N-Tertiarybutyloxycarbonyl-4-(N-hydroxysuccinimidocarbonyl)benzoylhydrazide

4 g (14.29 mmol) N-tertiarybutyloxycarbonyl-4-carboxybenzoylhydrazideare dissolved together with 3.29 g (28.6 mmol) of N-hydroxysuccinimideand 17.4 mg of DMAP in 100 ml of THF and diluted drop-wise at 4° C. witha solution of 3.23 g (15.72 mmol) of DCC. After 12 h of stirring at 4°C. and 48 h at room temperature, the solvent is removed under a vacuum,the residue dissolved in ethylacetate and extracted 10 times each with50 ml of saturated NaCl solution. After drying of the organic phase overNa₂SO₄, it is concentrated to half and cooled to 4° C. Colorless needlesare formed which are suctioned off and washed with a small amount ofdiethylether. Obtained are 5.3 g (14.09 mmol), corresponding to 98.6% ofthe theoretically possible yield. C₁₇H₁₈N₃O₇ (376 g/mol); calculated: C:54.26% H: 4.79% N: 11.18%; found: C: 52.65% H: 5.53% N: 10.56%

4-(N-hydroxysuccinimido)carbonyl)benzoylhydrazide trifluoroacetate

3 g (7.98 mmol) ofN-tertiarybutyloxycarbonyl-4-(N-hydroxysuccinimidocarbonyl)benzoylhydrazideare diluted with 10 ml of anhydrous trifluoroacetic acid and stirredover a period of 2 h at room temperature. During rigorous stirring, 50ml of diethylether is added. The solid formed is suctioned off andwashed several times with diethylether. After drying under a vacuum, 3.0g (7.67 mmol) of the product are obtained as a white powder,corresponding to 96.1% of the theoretically possible yield. C₁₄H₁₂N₃O₇F₃(391 g/mol); calculated: C: 42.97% H: 3.07% N: 10.74%; found: C: 43.49%H: 3.42% N: 10.74%

Step 2: Synthesis of Maleinimide and Hydroxysuccinimide Derivatives ofCytostatic Compounds

Chloroambucilcarboxylic acid hydrazide

Chloroambucil (1.0 g; 3.29 mmol) was dissolved in 50 ml of absoluteCH₂Cl₂ and diluted with oxalylchloride (431 μl; 4.8 mmol) and the set-upstirred for 15 h at 30-40° C. Subsequently, the solution is concentratedthrough evaporation and residues of oxalylchloride removed under a highvacuum. The synthesized oxalylcarboxyl acid chloride was directlyreacted further by dissolving the obtained brown syrup in 20 ml ofabsolute CH₂Cl₂ and, while stirring at room temperature,tert.-butylcarbazate (457 mg; 3.45 mmol), dissolved in 20 ml of CH₂Cl₂,was added drop-wise within 1 h. The set-up was stirred for 36 h,filtered off from the insoluble parts and the solution concentratedunder a vacuum to approximately 3 ml. The obtainedchloroambucil-tert.-butylcarbazate was brown syrup purified using acolumn chromatography (running medium: acetic ester/hexane 2/1;R_(f)-value: 0.52); yield: 700 mg (1.67 mmol; 51% of the theoreticalvalue). Chloroambucil-tert.-butylcarbazate (700 mg; 1.67 mmol) wasdissolved in 10 ml of tetrahydrofuran and diluted while stirring at roomtemperature with 10 ml of trifluoroacetic acid, stirred for 1 h andsubsequently the solvent and the trifluoroacetic acid removed under ahigh vacuum with the formation of a light-brown solid.

Carboxylhydrazone derivative of[4-(4-bis(2-chloroethyl)aminophenyl)]butyric acid hydrazide(trifluoroacetate salt) and 4-acetophenonecarboxylicacid-(N-hydroxysuccinimide)-ester

Chloroambcilcarboxylic acid hydrazide (trifluoroacetate salt; 721 mg;1.67 mmol) was dissolved in 30 ml of tetrahydrofuran and diluted whilestirring at room temperature with 4-acetophenonecarboxylicacid-(N-hydroxysuccinimide)-ester (479 mg; 1.83 mmol). After 20 minutes,the reaction solution was concentrated by evaporation. The residue wasdissolved and recrystallized out from acetic ester/hexane; yield: 290 mg(0.66 mmol; 40% of the theory) light-yellow crystals; (C₂₇H₃₁N₄O₆Cl₂, Mr577),

calculated C: 41.25% H: 5.4% N: 9.7% Cl: 12.1% found C: 41.11% H: 5.6%N: 12.4% Cl: 11.7%

N¹-(2-Maleinimidoethyloxycarbonyl)-5-fluorouracil

500 mg (3.84 mmol) 5-fluorouracil were dissolved in 100 ml oftetrahydrofuran and diluted with 505 mg (696 μl; 4.99 mmol) oftriethylamine. To this solution, 1016 mg (4.99 mmol) of2-maleinimidoethyl chloroformate, dissolved in 100 ml oftetrahydrofuran, were added drop-wise and the set-up was stirred for 15h at room temperature. Then, the solvent was removed under the water jetvacuum and the residue purified using column chromatography(LOBAR®-column; running medium: acetic acid/hexane 1.5/1; yield: 61%);melting point: 132° C., R_(f)-value: 0.64 (diol; acetic acid/hexane 2/1)

calculated C: 44.29% H: 2.68% N: 14.09% (C₁₁H₈N₃O₆F); found C: 44.29% H:2.68% N: 13.58%

N¹-(m-Maleinimidobenzoyloxymethyl)-5-fluorouracil

1500 mg (11.55 mmol) 5-fluorouracil and 2.25 ml (25.4 mmol) of 37-%formaldehyde solution were heated to 60° C. while stirring for 2 h.Then, the water was removed under a high vacuum and the residuedissolved with the addition of 20 mg of DMAP in 80 ml oftetrahydrofuran. To this solution, 3.01 g (13.85 mmol) ofm-maleinimidobenzoic acid and 2.858 g (13.85) of DCC, dissolved in 50 mlof tetrahydrofuran, were added and then stirred for 15 h at roomtemperature. The precipitate was filtered off and the solvent removedunder a vacuum. The residue was taken up in approximately 20 ml ofacetic acid and filtered off from insoluble parts. The solution wasconcentrated by evaporation and the residue chromatographed (1. silicagel (acetic ester/hexane 2/1); yield: 5% white powder); melting point:decomposition>250° C.; R_(f)-value: 0.32 (acetic acid/hexane 2/1)

Elementary analysis calculated: C: 55.98% H: 2.92% N: 12.24%;(C₁₆H₁₀N₃O₆F) found: C: 55.51% H: 2.87% N: 11.72%

3′-Aminoamide derivative of doxorubicine with p-maleinimidophenylaceticacid chloride

500 mg (0.86 mmol) doxorubicine hydrochloride were dissolved in 50 ml ofabsolute DMF and 1013 mg (4.30 mmol) of p-maleinimidophenylacetic acidchloride and 719 μl (522 mg; 5.16 mmol) of triethylamine were added. Thesolution was stirred at room temperature for 15 h. DMF was removed undera high vacuum and the residue was dissolved in 5 ml of tetrahydrofuran,filtered and purified over a silica gel column (tetrahydrofuran/hexane3/1); 189 mg of the red product (29%); R_(f)-value: 0.26(ethylacetate/hexane 3/1); melting point: 110° C.; (C₃₉H₃₆N₂O₁₄);

calculated C: 61.84% H: 4.75% N: 3.70%; found C: 61.35% H: 5.14% N:3.45%.

C-13-Benzoylhydrazone derivative of doxorubicine and4-((N-hydroxysuccinimido)carbonyl)benzoylhydrazide trifluoroacetate

0.2 mmol doxorubicine hydrochloride and 1.0 mmol and4-((N-hydroxysuccinimido)carbonyl)benzoylhydrazide trifluoroacetate saltwere dissolved in 100 ml of methanol. To this solution, 100 μl ofCF₃COOH were added and the reaction mixture stirred for 36 h at roomtemperature. The solution was then concentrated to approximately 50 ml.Acetonitril was added until reaching turbidity and the suspension wascooled at −20° C. for 24 h. The product was collected by means ofcentrifugation, and dissolved and recrystallized frommethanol/acetonitril: 276 mg; R_(f)-value (reverse phase,acetonitril/0.005 M NaH₂PO₄ (pH 5.0)=70/30): 0.33, melting point: >250°C. (decomposition), (C₃₉H₄₀N₄O₁₄Cl)

calculated C: 56.83% H: 4.86% N: 6.80% Cl: 4.31%; found C: 57.04% H:5.14% N: 6.55% Cl: 4.12%

The method for the production of maleinimide derivatives withcis-configured platinum(II) units is described by the following example:N—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-1,2-diaminoethanedichloroplatinum(II)—four-step synthesis.

N-(2-Hydroxyethyl)-N,N′-bis-tertiarybutyloxycarbonyl-1,2-diaminoethane

To a solution of 20.8 g (200 mmol) ofN-(2-hydroxyethyl)-1,2-diaminoethane in 100 ml of dichloromethane, 47.96g (220 mmol, 0.5 eq) of bis-tertiarybutyloxycarbonylanhydride, dissolvedin 200 ml of dichloromethane, were added drop-wise within 1 h at roomtemperature. The set-up is stirred for 12 h at room temperature, thendiluted with 100 ml of diethylether and extracted with 150 ml of water.After drying of the organic phase over sodium sulfate, it isconcentrated under a vacuum. The purification of the product occursthrough the use of column chromatography (ethylacetate/hexane (1:1.5),R_(f)-value: (ethylacetate/hexane 1:1.5): R_(f)=0.18; yield: 30 g (98.68mmol). Elementary analysis for C₁₄H₂₈N₂O₅ (304 g/mol); calculated: C:55.26% H: 9.21% N: 9.21%; found: C: 55.50% H: 9.18% N: 9.02%

N—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-N,N′-bis-tertiarybutyloxycarbonyl-1,2-diaminoethane

To a solution of 8 g (26.3 mmol)N-(2-hydroxyethyl)-N,N′-bis-tertiarybutyloxycarbonyl-1,2-diaminoethanein 50 ml of tetrahydrofuran and 4 ml (28.9 mmol, 1.1 eq) oftriethylamine, 6.82 g (29 mmol, 1.1 eq) of maleinimidobenzoic acidchloride, dissolved in 100 ml of tetrahydrofuran, are added drop-wise atroom temperature while stirring within 1 h. After stirring for a further8 h at room temperature, according to DC, the reaction is completed. Thetriethylammonium chloride formed in the reaction is filtered off. Afterremoval of the tetrahydrofuran and the excess triethylamine under avacuum, the oil formed is purified using column chromatography (silicagel: ethyl acetate/hexane (1:1), R_(f)-value (ethyl acetate/hexane1:1)=0.2, yield: 9.6 g (19.1 mmol) of the product in the form a yellowoil, corresponding to 72.6% of the theoretically possible yield.Elementary analysis for C₂₅H₃₃N₃O₈ (503 g/mol); calculated: C: 59.64% H:6.56% N: 8.35%; found: C: 60.04% H: 6.77% N: 8.24%

N—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-1,2-diaminoethanedihydrochloride

6 g (11.93 mmol) ofN—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-N,N′-bis-tertiarybutyloxycarbonyl-1,2-diaminoethaneare diluted with 60 ml (5 eq) of a 1M solution of HCl in diethyletherwhile stirring at room temperature. After 48 h of stirring at roomtemperature, the fine-crystallinic precipitate is suctioned off over aG4 glass frit, freed of the HCl residues through multiple washing withanhydrous ether and dried under a vacuum. Obtained are 3.0 g (7.98 mmol)of the product as a fine-powdery yellow solid, corresponding to 66.9% ofthe theoretically possible yield. Elementary analysis for C₁₅H₁₉N₃O₄Cl₂(375.9 g/mol);

calculated C: 47.89% H: 5.05% N: 11.17% Cl: 18.86%; found C: 46.97% H:5.42% N: 10.03% Cl: 17.63%

N—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-1,2-diaminoethanedichloroplatinum(II)

104 mg (0.25 mmol) of K₂PtCl₄ and 94.2 mg (0.25 mmol) ofN—(O—(3-maleinimidobenzoyl)-2-diaminoethane dihydrochloride aredissolved in 5 ml each of 20% tetrahydrofuran/water and mixedportion-wise. The initially red K₂PtCl₄ solution is increasinglydecolorized; a light-yellow precipitate is formed. Approximately 1 hafter the last addition can the precipitate be suctioned off using a G4glass frit. It is washed successively with small amounts of water, 20%tetrahydrofuran/water and finally with diethylether. After drying undera vacuum, obtained are 106 mg (0.18 mmol) of the product as afine-crystalline substance, corresponding to 72% of the theoreticallypossible yield.

Elementary analysis for C₁₅H₁₇N₃O₄PtCl₂ (569 g/mol); calculated: C:31.63%, H: 2.99%, N: 7.38%, Pt: 34.29, Cl: 12.46%; found: C: 31.19%, H:3.37%, N: 6.79%, Pt: 32.35%, Cl: 12.95%

Step 3: Thiolation of the Carrier Protein

The method for the thiolation is illustrated in more detail through thefollowing example: 32 mg of human serum transferrin (98% cyrstalline, Mr80,000) [or 28.4 mg of human serum albumin (98% crystalline, Mr 66500),respectively] are dissolved in 1 ml of buffer (0.1 M sodium borate,0.001 M EDTA, 0.15 M NaCl—pH=8.0) degassed with argon(c(transferrin/albumin)=4.0×10⁻⁴ M) and diluted with 100 μl of a freshlyproduced 4.0×10⁻² M iminothiolane solution (5.5 mg of iminothiolanedissolved in 1 ml of degassed buffer (0.1 M sodium borate, 0.15 M NaCl,0.001 M EDTA—pH=8.0). After 60-70 minutes, the excess iminothiolane isseparated through gel filtration (column 1.0 cm×10 cm, Sephadex G.25)with the running buffer of 0.05 M sodium borate, 0.15 M NaCl—pH 7.5 ofthiolated transferrin or albumin. The protein concentration aftercompleted gel filtration was was determined at 280 nm ε₂₈₀=92300 M⁻¹cm⁻¹, c[transferrin]=2.4×10⁻⁴ M) or ε₂₈₀=35700 M⁻¹ cm⁻¹,(c[albumin]=2.2×10⁻⁴ M) and the number of the introduced HS groups wasdetermined with Ellmanns reagent at 412 nm ε₄₁₂=13600 M⁻¹ cm⁻¹) (c[HSgroup]=7.4×10⁻⁴ M or 7.7×10⁻⁴ M). The ratio c[HS groups]/c[transferrin]was thus 3.1 and the ratio c[HS groups]/c[albumin] was 3.5. Thefollowing tables summarize the reaction conditions, by which differentnumbers of HS groups are introduced into transferrin or albumin:

TABLE 1a Reaction conditions for the introduction of HS groups intotransferrin c[iminothiolane]/ reaction number of introduced c[protein]time, min temperature, ° C. HS groups/protein 10:1 60-75 0-5 1 10:160-75 20-25 2-3 20:1 60-75 20-25 5-6 30:1 60-75 20-25  9-10 40:1 60-7520-25 12-13 50:1 60-90 20-25 15-16 70:1 60-90 20-25 20-21

TABLE 1b Reaction conditions for the introduction of HS groups inalbumin Reaction conditions for the introduction of HS gronps intotransferrin c[iminothiolane]/ reaction number of introduced c[protein]time, min temperature, ° C. HS groups/protein 10:1 60-75 0-5 1 10:160-75 20-25 2-3 20:1 60-75 20-25 5-6 30:1 60-75 20-25  9-10 50:1 60-9520-25 15-16 70:1 60-90 20-25 30-32

The thus isolated protein sample was used directly for the followingreaction in Step 4.

Step 4: Coupling of the Derivatives of the Cytostatic Compounds (Step 2)to the Thiolated Carrier Protein (Step 3) or to Polyethylene Glycols:

Methods—FPLC for the production of conjugates: P-500 pump, LCC 501controller (Pharmacia) and LKB 2151 uv-monitor, buffer: standard borate:0.025 M sodium borate, 0.15 M NaCl—pH 7.5 or phosphate buffer: 0.004 Msodium phosphate, 0.15 M NaCl—pH 7.4. The protein concentration of theconjugate was determined with the BCA protein essay from Pierce (USA).

Transferrin conjugate with the 3′-amino amide derivative of doxorubicineand p-maleinimidophenylacetic acid (amide₁)

3.5 ml of thiolated transferrin sample (3.3 introduced HS groups) werediluted to 30 ml with standard borate and 1.0 ml of a solution of amide₁(Mr 742.68) in DMF (1.8 mg dissolved in 1.0 ml of DMF) were added andmixed. After 10 min, the solution was concentrated to approximately 2.0ml with CENTRIPREP®-10 concentrators from Amicon, FRG (60 min at 4° C.and 4500 U/min). The concentrated sample was centrifuged (5 min) with aSigma 112 centrifuge and the excess applied on a Sephadex® G-25F column(column 1.0 cm×10 cm) and the conjugate was isolated (retention volume:3.5-7.0 ml). The amount of bound doxorubicine was determined with theaid of epsilon values for doxorubicine ε₄₉₅=10650 M⁻¹ cm⁻¹, from whichthe corresponding contribution of transferrin at this wavelength wassubtracted ε[Tf]₄₉₅=4100 M⁻¹ cm⁻¹. The concentration of the bounddoxorubicin was 322 μM and that of transferrin was 101 μM.

Albumin conjugate of the carboxyl hydrazone derivative of[4-(4-bis(2-chloroethyl)amino-phenyl)]butyric acid hydrazide and4-acetophenone carboxylic acid-(N-hydroxysuccinimide)-ester

66.5 mg of albumin, dissolved in 2.2 ml of 5 ml phosphate buffer, weremixed with 0.1 ml of a solution of carboxylhydrazone derivative in DMF(1.2 mg dissolved in 0.1 ml of DMF), centrifuged after 10 min and theexcess apllied on a Sephadex® G-25F column (column 1.0 cm×10 cm) and theconjugate was isolated (retention volume: 3.7-7.1 ml). The amount ofbound chloroambucil was determined with the aid of the test according toEpstein (Epstein et al., Anal. Chem. 1955, 27, 1435-1439). It was 280 μMin this working method.

Polyethylene glycol conjugate consisting of CH₃O-PEG-SH 20,000(α-methoxy-ω-thio-polyethylene glycol) andN—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-1,2-diaminoethanedichloroplatinum(II)

100 mg of CH₃O-PEG-SH 20,000 (0.005 mmol) are dissolved in 5 ml ofphosphate buffer and mixed with 5.7 mg (0.01 mmol) ofN—(O—(3-maleinimidobenzoyl)-2-hydroxyethyl)-1,2-diaminoethanedichloroplatinum(II), dissolved in 250 μl of DMF. After 20 min, thereaction mixture was centrifuged and the excess applied on a Sephadex®G-25F column (column 2.0 cm×15 cm) and the conjugate was isolated. Theamount of bound platinum complex was determined with the aid of the testaccording to Gonias and Pizzo (Gonias, S. L., Pizzo, S. V. J.Biol.Chem.1982, 258, 5764-5769). It was 380 μM in this working method.

Polyethylene glycol conjugate consisting of HS-PEG-SH 20,000(α,ω-bis-thio-polyethylene glycol) andN′-(2-maleinimidoethyloxycarbonyl)-5-fluorouracil

100 mg of HS-PEG-SH 20,000 (0.005 mmol) are dissolved in 5 ml ofphosphate buffer and mixed with 3.0 mg (0.01 mmol) ofN′-(2-maleinimidoethyloxycarbonyl)-5-fluorouracil, dissolved in 250 μlof DMF. After 30 min, the reaction mixture was centrifuged and theexcess applied on a Sephadex® G-25F column (column 2.0 cm×15 cm) and theconjugate was isolated. The amount of bound 5-fluorouracil wasdetermined at ε₂₆₀=6520 M⁻¹ cm⁻¹. It was 460 μM in this working method.

Conjugate consisting of albumin and transferrin loaded with doxorubicin(amide₁)

30 mg of human serum albumin were dissolved in 2.0 ml of 0.1 M sodiumborate degassed with argon, 0.001 M EDTA, 0.15 M NaCl—pH=8.0 at roomtemperature (RT). To this mixture, 183 μl of a solution of 2.9 mg ofiminothiolane in 250 μl of degassed 0.1 M sodium borate, 0.001 M EDTA,0.15 M NaCl—pH=8.0 were added, thoroughly mixed and incubated for 1 h atroom temperature. Then, the excess iminothiolane was separated offthrough gel filtration. The measurement of protein concentration, aswell as the concentration of free SH groups resulted in the followingvalues: c[albumin]=1.44×10⁻⁴ mol/l, c[SH groups]=1.25×10⁻³ mol/l. Theaverage number of SH groups per albumin molecule resulted from thequotient of both concentrations to be 8.7.

2.0 ml of this thiolated albumin solution were used for the furtherreaction. To neutralize 7 of the 8.7 SH groups per protein with3′-aminoamide derivative of doxorubicin and p-maleinimidophenylaceticacid (amide₁), the protein solution was first diluted with standardborate buffer to 10 ml. Then, while shaking, 40 μl of a solution of 40mg of amide₁ in 1 ml of DMF was added and incubated for 10 minutes atroom temperature. After that, the sample was centrifuged (3,000 g, 4°C.) for 5 min. The excess was concentrated to a volume of approximately1 ml (3,000 g, 3×15 min, 4° C.) using Centricon 3,000 concentrators®(company: Amicon). Then, 25 mg of human serum transferrin was dissolvedin 1.2 ml of buffer (0.1 M sodium borate, 0.001 M EDTA, 0.15 MNaCl—pH=8.0) degassed with argon. To this mixture, 15 μl of a solutionof 2.9 mg of iminothiolane in a 250 μl of buffer (0.1 M sodium borate,0.001 M EDTA, 0.15 M NaCl—pH=8.0) degassed with argon were added,thoroughly mixed and incubated for 1 h at room temperature. Then, excessiminothiolane was separated off through gel filtration. The measurementof protein concentration as well as the concentration of free SH groups(test according to Ellmann) resulted in the following values:[transferrin]=1.20×10⁻⁴ mol/l, [SH groups]=1.96×10⁻⁴ mol/l. The averagenumber of SH groups per transferrin molecule resulted from the quotientof both concentrations to be 1.6. 1.9 ml of the thus-thiolatedtransferrin solution were diluted to 10 ml for the further reaction,first, with standard borate buffer. Then, to the reaction mixture, 72 μlof a solution of 10 mg of 1,4-diamino-N,N′-di-m-maleinimidobenzyl-butanein 200 μl of DMF were added. After 10 min at room temperature, theturbid mixture was centrifuged (5 min, 3,000 g, 4° C.). The excesssolution was poured off and concentrated in a Centricon 3,000concentrator® to a volume of 600 μl. For removing of the excessbismaleinimide, it was filtered over a Sephadex 25. 1,500 μl of asolution with the thus-modified transferrin were added to 500 μl of theabove albumin solution loaded with doxorubicin, mixed thoroughly andincubated for 15 min at room temperature. Thereafter, the solution wasconcentrated to a volume of 150 μl in Microcon 10 concentrators®(company: Amicon). The concentrated protein solution was separatedthrough gel chromatography into its ingredients (monomers, dimers,oligomers). Dimensions of the column: h: 40 cm, ø: 1 cm, loop: 100 μl,stationary phase: Superdex 200 Pharmacia, mobil phase: borate buffer, pH6.8; gassed at +4° C. with N₂, flow: 1 ml/min, detection: photometric,λ=280 nm, retention volume: oligomers: 9.5 ml-11.5 ml, trimers: 11.7ml-12.6 ml, dimers: 12.7 ml-14.4 ml, monomers: 14.5 ml-18.5 ml.

The yield of the desired dimers with regard to the introduced amide₁ was20-30%.

Biological Studies

As an example for the in vitro and in vivo effectiveness of theconjugates, given are biological data of the doxorubicin conjugatesshown below, which are representative of the activity- or toxicityprofile of the conjugates disclosed in the invention. The effectivenessof the conjugates was determined using a “colony-forming assay” and withthe aid of the incorporation of BrdU(5-bromo-2-desoxyuridine) in thecell culture according to standard scientific practice. As examples,shown are the IC₇₀ values (“colony-forming assay”—seven xenografts) ofthe following doxorubicin conjugates: protein=transferrin (T) or albumin(A)

IC70 values in seven human tumor xenografts (colony-forming assay) humantumor T-DOXO- T-DOXO- DOXO- A-DOXO- A-DOXO- xenograft HYD ARZID RUBICINARZID HYD bladder BXF 1.48 0.09 0.50 0.06 0.10 1299 lung LXFL 0.054 0.060.02 0.03 0.04 529 lung LXFS 0.04 0.04 0.02 0.00 1 0.01 538 mamma 0.040.04 0.02 0.01 0.10 MAXFMX1 melanoma 0.29 0.23 0.30 0.34 0.30 MEXF9S9prostate 0.02 0.03 0.03 0.02 0.06 PC3MX prostate 0.12 0.08 0.35 0.240.21 DU145X

Under these experimental conditions, the synthesized conjugates aslisted above showed an effectiveness that was equal to or higher thanthat of the unbound cytostatic compounds. Corresponding polyethyleneglycol conjugates exhibited a similar behavior.

Furthermore, the above-shown conjugates exhibit, in comparison to thefree doxorubicin, in the xeno-transplantable naked-mouse models, mammarycarcinoma MDA-MB-435 and mammary carcinoma MCF-7, in total a clearlyreduced toxicity (reduced lethalness and body weight reduction, fewerside-effects in the gastro-intestine area), as compared to the freedoxorubicin, and a stabilization of the increase in the relative tumorvolume with equal or improved tumor-inhibiting effectiveness, as isshown in the example of the transferrin conjugate T-DOXO-HYD in thetable:

Animals: Ncr:nu/nu female; tumor: mammary carcinoma MCF-7 s.c. Therapy:day (d) 16, day (d) 23 i.v. body weight loss number dose mortality (%) doptimum of mice substance (mg/kg/inj.) (d) 13-23 TIC (%) 8 NaCl 4 8doxorubicin 4 −5 61 8 doxorubicin 8 1 (26) −15 39 7 doxorubicin 12  7(25-33) −21 8 T-DOXO-HYD 4 −4 82 8 T-DOXO-HYD 8 −4 42 8 T-DOXO-HYD 12 −6 21

The dose refers to the amount of doxorubicin available. In the case ofan equimolar dose (8 mg/kg), T-DOXO-HYD exhibits an effectiveness thatis comparable to that of doxorubicin at reduced lethalness and bodyweight reduction. In the case of the highest dose employed in theexperiment (12 mg/kg), there appears a very high lethalness (5 of 7 and7 of 7 animals, respectively) in the therapy with doxorubicin. Thetherapy with the transferrin conjugate at this dose exhibits nolethalness, the anti-tumor effectiveness being better.

What is claimed is:
 1. A conjugate of a cytostatic compound and apolyethylene glycol, wherein: said cytostatic compound is coupled via aspacer comprising a group which is derived from a maleinimido group, orto polyethylene glycol having at least one HS or H₂N group; and whereinsaid polyethylene glycol has a mass of about between 5,000 and 200,000Da.
 2. The conjugate according to claim 1, wherein said cytostaticcompound is selected from the group consisting of anthracyclines,nitrogen mustard gas derivatives, purine or pyrimidine antagonists,folic acid antagonists, taxoids, camptothecines, podophyllotoxinderivatives, vinca alkaloids and cis-configured platinum(II)-complexes.3. The conjugate according to claim 1, wherein said cytostatic compoundis selected from the group consisting of doxorubicine, daunorubicine,epirubicine, idarubicine, mitoxandrone, chloroambucil, melphalan,5-fluorouracil, 5′-deoxy-5-fluorouridine, thioguanine, methotrexate,paclitaxel, docetaxel, topotecane, 9-aminocamptothecine, etoposide,teniposide, mitopodozide, vinblastine, vincristine, vindesine,vinorelbine and compounds of the general formulas I, II, III or IV:

wherein n=0-6, X=—NH₂, —OH, —COOH, —O—CO—R—COR* or —NH—CO—R—COR*,wherein R is an aliphatic carbon chain having 1-6 carbon atoms or is asubstituted or unsubstituted phenylene group and R* is H, phenyl oralkyl having 1-6 carbon atoms.
 4. The conjugate according to claim 1,wherein said cytostatic compound having said spacer comprising a groupwhich is derived from a maleinimido group, is formed through reaction ofsaid cytostatic compound with a maleinimide compound of the formula V,VI or VII:

where R is an aliphatic carbon chain having 1-6 carbon atoms or asubstituted or unsubstituted benzyl group or a substituted orunsubstituted phenylene group, Y=—OH, —COOH, —COCl, —CONH—(CH₂)_(n)—OH,—COO—(CH₂)_(n)—NH₂, —COO—(CH₂)_(n)—NHNH₂, —SO₃H, —SO₃Cl, —SO₂—NHNH-₂,—O—COCl, —CHO or —COR*, wherein n=1-6 and R* represents H, phenyl oralkyl having 1-6 carbon atoms, and the thus obtained maleinimidederivative of said cytostatic compound is coupled to said polyethyleneglycol, wherein the chemical linkage between said cytostatic compoundand said maleinimide compound occurs through an amide, ester, imine,hydrazone, carboxyl hydrazone, oxycarbonyl, acetal or ketal bond.
 5. Amethod for the production of a conjugate of a polyethylene glycolaccording to claim 1, comprising the steps of: (a) reacting a cytostaticcompound with a maleinimide compound, such that maleinimide derivativesof said cytostatic compound are produced, wherein the chemical linkagebetween said cytostatic compound and said maleinimide compound occursthrough an amide, ester, imine, hydrazone, carboxyl hydrazone,oxycarbonyl, acetal or ketal bond; and (b) coupling said maleinimidederivative obtained in step (a) of the cytostatic compound topolyethylene glycol having at least one HS or H₂N group and having amass of about between 5,000 and 200,000 Da.
 6. A pharmaceuticalcomposition, comprising the conjugate according to claim 1, optionallytogether with carriers and auxiliary agents.
 7. Method for the treatmentof a cancer disease, comprising the step of treating an organism havinga cancer disease with the conjugate of claim
 1. 8. Method according toclaim 7, wherein said cancer disease comprises bladder, lung, mamma,melanoma or prostrate carcinomas.